The cAMP-specific cyclic nucleotide phosphodiesterases (PDE4 enzymes) are a diverse family of enzymes that control degradation of the second messenger cAMP and thus are important regulators of intracellular signaling. They are also the specific targets for an important class of drugs with therapeutic potential in humans, especially in disorders of the central nervous system and of the immune/inflammatory systems, such as asthma. Long-Term Goals: To determine which cellular signaling pathways regulate the PDE4 enzymes, as follows: 1. To characterize the regulation of the PDE4 enzymes by novel protein-protein interactions. 2. To characterize the regulation of the PDE4 enzymes by novel phosphorylation events. Specific Aims: 1. To characterize the interactions between the human cAMP-specific PDE4D5 isoform and the RACK1 anchoring protein. 2. To characterize the regulation of the cAMP-specific PDE4A5 isoform by the AIP/ARA9 protein, a newly identified immunophilin. 3. To characterize the phosphorylation of the cAMP-specific PDE4A5 isoform by three different kinases: cAMP- dependent protein kinase (PKA), MAPK and p70rsk. 4. To isolate novel cDNAs that encode cAMP-specific PDEs, derived from transcripts in the mammalian brain and olfactory system. 5. To isolate cDNAs enoding novel proteins that interact with additional human cAMP-specific PDE isoforms. Research Plan and Methods: 1. The ability of RACK1 and PDE4D5 to interact in mammalian cells will be tested by co- immunoprecipitation. To determine the site(s) on each protein responsible for their interaction, mutations in RACK1 or PDE4D5 will be tested for interaction using the two-hybrid system and by expression in mammalian cells. The effect of these mutations on the targeting of PDE4D5 to membranes will also be tested. 2. To test for an interaction between PDE4A5 and ARA9/AIP, they will be co- immunoprecipitated from cells. Mutant forms of the PDE4A5 isoform and the ARA9/AIP protein will also be tested for their interaction in cells. The interaction between bacterially-expressed proteins will also be studied. Interactions between ARA9/AIP and p70rsk and other kinases will be tested by co-immunoprecipitation. 3. Site-directed mutagenesis will be used to create mutations at putative MAPK, p70rsk and PKA sites in the PDE4A5 protein. These will be expressed in mammalian cells and assayed for changes in enzymatic activity. The phosphorylation status of PDE4A5 isolated from cells will also be studied. 4. cDNA libraries from brain and olfactory tissue will be screened for new PDE4 isoforms, and their tissue distribution and enzymatic properties will be determined. 5. The two-hybrid system will be used to clone cDNAs encoding proteins that interact with 4 novel PDE4 isoforms.